Lentiviral-based vectors and related systems and methods for eukaryotic gene editing

A lentiviral packaging system comprising:

• a) a packaging plasmid comprising a eukaryotic promoter operably linked to a Gag nucleotide sequence, wherein the Gag nucleotide sequence comprises a nucleocapsid (NC) coding sequence and a matrix protein (MA) coding sequence, wherein one or both of the NC coding sequence or the MA coding sequence comprises at least one non-viral aptamer-binding protein (ABP) nucleotide sequence, and wherein the packaging plasmid does not encode a functional integrase protein;
• and
• b)
• (i) a mammalian expression plasmid comprising a eukaryotic promoter operably linked to a non-viral nucleic acid sequence, wherein the non-viral nucleic acid sequence comprises a CRISPR-associated endonuclease coding sequence and a guide RNA (gRNA) coding sequence, wherein the gRNA coding sequence comprises at least one aptamer coding sequence;
• or
• (ii) two mammalian expression plasmids wherein each plasmid comprises a eukaryotic promoter operably linked to a non-viral nucleic acid sequence;
• wherein the non-viral nucleic acid sequence in the first plasmid comprises a CRISPR-associated endonuclease coding sequence, wherein the non-viral nucleic acid sequence in the second plasmid comprises a guide RNA (gRNA) coding sequence, and wherein the gRNA coding sequence comprises at least one aptamer coding sequence.